Vasculogenesis begins early in vertebrate development and culminates in the formation of a complex network of arteries, veins, and capillaries. Once formed, the gross and microscopic structure of this network is stable unless disrupted by disease. Genetic and cell culture studies have begun to identify molecular determinants of vasculogenesis, and these determinants have defined three distinct stages of vascular development (Hanahan, 1997; Folkman and D'Amore, 1996). In the first stage, splanchnic mesoderm coalesces to form simple tubes of endothelial cells. Vascular endothelial growth factor defines this stage (Shalaby et al., 1995; Fong et al., 1995; Carmeliet et al., 1996). The second stage involves the recruitment of mesenchymal cells by the endothelium, a process coordinated by angiopoietin and platelet-derived growth factor (Ferrara et al., 1996; Suri et al., 1996; Lindahl et al., 1997; Sato et al., 1995). In the third stage, mesenchyme differentiates into smooth muscle and extracellular matrix deposition begins. Transforming growth factor beta has been implicated in this stage (Folkman and D—Amore, 1996; Beck and D'Amore, 1997). After the third stage of vascular development, arterial smooth muscle cells exit the cell cycle and vascular structure is stabilized (Schwartz et al., 1990; Owens, 1995; Glukhova et al., 1991). The molecular determinants of this final stage are unknown.
There is growing evidence that the extracellular matrix regulates cellular function during organogenesis. Fibronectin, vitronectin, collagen, and other extracellular matrix proteins bind to integrins on the surface of cells (Gumbiner, 1996; Hynes, 1992), providing morphogenic signals that regulate cell proliferation, migration, and differentiation (Adams and Watt, 1993; Hynes, 1994). Disruption of fibronectin in mice causes dramatic developmental abnormalities, including failure to develop a notochord and somites (George et al., 1993). Null mutations in genes encoding fibronectin receptors, or integrins, lead to embryonic or perinatal death from developmental abnormalities resembling those observed in mice lacking fibronectin (Yang et al., 1993; Yang et al., 1995). Not all cell-matrix interactions, however, are necessary for normal morphogenesis. For example, disruption of vitronectin, tenascin C, and integrin alpha 1 have no apparent effect on development (Zheng et al., 1995; Saga et al., 1992; Gardner et al., 1996).
Elastin is the dominant arterial extracellular matrix protein (Parks et al., 1993). This protein is encoded by a single gene, and organized into polymers that form concentric rings of elastic lamellae around the arterial lumen. Each elastic lamella alternates with a ring of smooth muscle, forming a lamellar unit. The function of elastic fibers was thought to be purely structural, providing tensile strength and resiliency to the aorta and other arteries. Because of its structural role, investigators believed that disruption of elastin would lead to dissection of arteries. This view was supported by studies associating decreased elastin content and increased elastase activity with arterial aneurysms in humans and other species (Thompson, 1996; Terpin and Roach, 1987). In addition, disruption of collagen I and fibrillin, prominent arterial extracellular matrix proteins, resulted in rupture of blood vessels in mice and humans (Lohler et al., 1984; Dietz and Pyeritz, 1995). Our human molecular genetic studies demonstrated, however, that ELN mutations do not cause arterial dilatation, but instead cause an obstructive arterial disease, supravalvular aortic stenosis (SVAS) (Curran et al., 1993; Ewart et al., 1993). To define the role of ELN in arterial development and disease, we generated mice hemizygous (ELN +/−) and homozygous (ELN −/−) for an ELN null mutation. From characterization of these mice, we conclude that elastin has two distinct functions. One is to provide arterial elasticity and ameliorate wall stress. A second is to control smooth muscle proliferation and organization during late arterial development. Elastin, therefore, is a molecular determinant of arterial morphogenesis, defining a fourth stage of development, the stage of arterial stabilization.
The publications and other materials used herein to illuminate the background of the invention or provide additional details respecting the practice, are incorporated by reference, and for convenience are respectively grouped in the appended List of References.